The present invention relates generally to methods and measuring instruments or sensors for use in biological, biochemical, and chemical testing, and particularly to methods, instruments, and the use of instruments which utilize surface plasmon resonance (SPR) for detecting molecules or monitoring structural and electronic changes in the molecules with ultra-high resolution and ultra-fast response times.
Surface plasmon resonance (SPR) is the oscillation of the plasma of free electrons which exists at a metal boundary. These oscillations are affected by the refractive index of the material adjacent the metal surface. It is this phenomenon that is used to detect minute changes in the refractive index of a surface and forms the basis of various sensor mechanisms. Surface plasmon resonance spectroscopy has emerged as a powerful technique in recent years for detection and analysis of chemical and biological substances in many research areas and industrial applications, such as surface science, biotechnology, environment, drug and food monitoring, and medicine. In biological sensors, detection of antibodies and their reactions with antigens using SPR is of primary interest in biomedical diagnostics, where the presence of antibodies associated with a bacteria or virus is an important indication of infection. SPR has also been applied to gene probes where deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) binding to a defined sequence in target analytes can be employed. In addition, SPR has found applications in detecting trace amount of toxic agents in air or in water for environmental protection or for chemical/biological warfare alert. Finally, SPR-based sensors are promising in food industry for detecting chemical and biological contamination in food. In all these application, improving the resolution and time response of SPR detection is of vital importance.
Surface plasmon resonance may be achieved by using the evanescent wave which is generated when a p-polarized light beam is totally internally reflected at the boundary of a medium, e.g. a glass prism, which has a high dielectric constant. A paper describing the technique has been published under the title xe2x80x9cSurface plasmon resonance for gas detection and biosensingxe2x80x9d by Lieberg, Nylander and Lundstrom in Sensors and Actuators, Vol. 4, page 299. The widely used methods for detecting SPR are based on attenuated total reflection (ART) of a collimated laser beam is incident on a glass body, usually a prism, on which a thin metal film is coated. When the incident light reaches an appropriate angle the reflection decreases sharply to a minimum, corresponding to the excitation of surface plasmon waves in the film. The total internal reflection is detected with a photodetector as a function of incident angle which is varied by rotating the prism. The photo detector is also rotated in order to catch the reflected light. When the incident beam reaches an appropriate angle, the reflection decreases sharply to a minimum that appears as a dip in the reflectivity vs. incident angle plot. The angular resolution achieved by this rotating prism approach is typically 10xe2x88x922-10xe2x88x923 deg (degrees), limited by errors in the angular position and noise in the intensity of the reflected light. For comparing different SPR detection techniques, the SPR resolution is often described in terms of the smallest detectable change in the refractive index for an analyte [refractive index units (RIU)]. The above angular resolution corresponds to 10xe2x88x925-10xe2x88x926 RIU at a wavelength of 630 nm. For higher angular resolutions, a large distance between the prism and the photodetector is required which makes the setup not only bulky but also more susceptible to mechanical noise and thermal drift. The response time is slow because of the mechanical movements in the setup.
Mechanical movements can be avoided by fixing the photodetector at an angle near resonance and measuring the intensity change in their reflection due to SPR angular shift. A major advantage of this approach is that the response time is only limited by the photodetector and the associated electronics which can be as fast as nanoseconds. A drawback, however, is that the relationship between the intensity and the sensitivity of the resonance angle measurement is dependent on the angle at which the photodetector is fixed. Major limitations in the resolution of the method come from fluctuation in the intensity of the laser and from thermal and mechanical drift in the setup.
Another widely use ATR-based method also fixes the position of the prism and replaces the collimated incident light in the above setups with a fixed convergent beam that covers a range of incident angles. This method is generally disclosed in xe2x80x9cThe ATR method with focused lightxe2x80x94application to guided waves on a gratingxe2x80x9d by E. Kretschmann, Vol. 26, number 1, Optics Communications, 1978, and in U.S. Pat. No. 4,997,278 by Finaln et. al. The reflections from different incident angles are collected simultaneously with a linear diode array (LDA) or charge coupled device (CCD) detector. This method involves no mechanical movements, but simultaneous detection of many channels (e.g., 1024 in a typical LDA) slows down the response time. The typical angular resolution is 10xe2x88x922-10xe2x88x923 deg or 10xe2x88x925-10xe2x88x926 RIU. As in the method with a rotating prism, high angular resolution of this method requires a large distance between the prism and the photodetector.
The above setups involve reflection intensity versus incident angle (an angle-scan system); SPR has also been detected by modulating the wavelength of incident light as described by Caruso, F., et al. (J. Appl. Phys., 1998, 83, 1023). The wavelength modulation causes modulation in the reflection intensity which is monitored with a lock-in amplifier and provides an accurate measurement of the SPR dip position. Using an acousto-optic tunable filter (AOTF), it was demonstrated that a wavelength change of 0.0005 nm, corresponding to 5xc3x9710xe2x88x927 RIU at a wavelength of 630 nm, can be detected. When applied to DNA-SH adsorption on gold, the signal to noise ratio of the AOTF SPR is six times better than that achieved by an angle-scan system.
As mentioned above, these methods suffer two major drawbacks: slow response time and limited angular resolution. The former one prevents the methods from detecting a fast process, such as the initial adsorption process of molecules onto surfaces, gas interactions, reactions between surface bound molecules and molecules in solution, and fast conformational changes in adsorbed proteins. The later one limits the sensitivity of SPR for detecting small amounts of molecules or small structural or conformational changes in molecules. In the first method, the response is slow because of mechanical movements involved in the method. The second method has no mechanical movements, but simultaneously detecting may channels (e.g., 1024 in a typical linear diode array) slows down the response time. For both methods, the angular resolution is typically poorer than 10xe2x88x923 degrees (typically on the order of 10xe2x88x922). For high angular resolution, both methods require a large distance between the sample and the detector, which makes the setups more susceptible to mechanical noise and thermal drift. Large distances, however, deteriorate the quality of the detected beam and makes to the SPR instruments bulky. For a given sample-detector distance, the resolution of the first method is limited by the precision of measuring the angular position of the prism. The resolution of the second method is limited by the number of channels (pixels) in the photo detector array and the noise level in the measured intensity in each channel. Improved resolution can be obtained using a software routine to fit the data collected by either the first or the second methods, however, this fitting procedure requires extra time and its reliability depends on the accuracy of each data point measured. The second method suffers an additional problem, in that the intensity of the beam is spread out over many channels, which decreases the signal to noise ratio, and therefore limits the resolution.
The present invention discloses a new SPR detection method that achieves an angular resolution in the order of 10xe2x88x925 deg (or 10xe2x88x928 RIU) and response times in the range of 1 xcexcs. The method has several additional features which include simplicity, good linearity, compactness, and immunity to ambient light. The method uses a convergent beam focused onto a thin metal film, but the total internal reflection is collected by a differential position or intensity sensitive photo-detecting device instead of a CCD or a LDA. The reflected light falling on the cell(s) of the differential position or intensity sensitive photo-detecting device is first balanced so that the SPR dip is located near the center of the differential position or intensity sensitive photo-detecting device. Because the differential signal is linearly proportional to the shift in the SPR angle and can be easily amplified without saturation problem, it provides an accurate detection of SPR. We note that a big-cell differential position sensitive photo-detecting device has been used by Alexander, S. Et al., (J. Appl. Phys. 1989, 65, 164) in the atomic force microscope (AM) in which the deflection of a laser beam due to bending of the AM cantilever is measured. In the present application it is the intensity distribution due to a SPR angular shift rather than physical movement of the laser beam that is measured.
The present method is carried out by focusing a diode laser through a prism onto a transparent plate coated with a thin metal film. The transparent plate is supported on an optical prism with index of refraction matching substance. The incident light and the differential position or intensity sensitive photo-detecting device are adjusted so that the SPR dip in the total internal reflection is located in the middle of the photo cells of the photodetecting system, corresponding to a zero differential signal from cell(s). On the metal film, a sample cell with necessary electrodes for controlling the electrochemical potential of the metal film is mounted into which molecules to be detected or studied or introduced. The presence of molecules or changes in the molecules on the metal film leads to a small shift in the SPR dip and results in a change in the differential signal of the differential position or intensity sensitive photo-detecting device that is easily amplified and detected.
One aspect of the present invention is to create a method of and sensor for detecting SPR for biological, biochemical, and chemical applications with a higher angular resolution and a faster response time. The angular resolution and speed of response is improved by being able to precisely position a differential position or intensity sensitive photodetecting device such that is centered on and detects the exact dip corresponding to surface plasmon resonance. Thus the detecting device can monitor changes with a response time of a few microseconds and angular resolutions on the order of 10xe2x88x925 degrees which is orders of magnitude better than previous methods.
An additional aspect of the present invention is to create a SPR sensor and detection method that is immune to ambient light, intensity fluctuations of the light source, and noise in the photo-detector and electronics.
A third aspect of the present invention is to modulate the SPR signal with the electrochemical potential of the metal film, for example using a lock-in technique, to improve the signal to noise ratio.
Another aspect of the present invention is to integrate electrochemical measurements, such as current, capacitance, and the like, into the SPR measurement, to provide important supplementary information about the detected molecules and improve specificity in the sensor and its applications. For example, since the ratio of the differential signal to the sum signal is proportional to the shift of the SPR angle, this method provides an accurate measurement of the SPR angle.
Yet another aspect of the present invention is to be able to miniaturize the SPR instruments and SPR-based sensors, which is important both for improving the thermal and mechanical stability of the instruments and sensor and for the convenience of using the instruments and sensors in a field environment. The SPR sensors based on the present invention are compact because they consist of only a focused light source, a prism and a photodetector, and high angular resolution is achieved without requiring a large sample-photodetector separation.
The above and other aspects, novel features, and advantages of the present invention will become apparent from the following detailed description of the preferred embodiments.